Bradykinin antagonist

ABSTRACT

A bradykinin antagonist of the formula: 
     
         (BKAn)(X)(Y) 
    
     where BKAn is a bradykinin antagonist peptide; Y is a pharmacore; and X is a bridging link chemically joining the BKAn and Y components.

RELATED APPLICATION

This is a continuation of application Ser. No. 08/296,185, filed Aug.29, 1994, which is an CON of 07/974,000, filed Nov. 10, 1992, nowabandoned which is a CIP of 07/859,582 filed Mar. 27, 1992 now abandonedand which is a CIP of 07/677,391 filed Apr. 1, 1991 now abandoned.

The present invention relates to pharmaceutically effective heterodimerscomprising a bradykinin antagonist (BKAn) component covalently linked toanother different pharmacophore component.

In the prior applications mentioned above, there are describedbradykinin antagonist dimers of the type:

    X(BKAn).sub.2

where BKAn represents a bradykinin antagonist peptide and X is a linkinggroup which joins the two BKAn components at points intermediate totheir ends. The BKAn substituents may be the same or different. However,also described are certain heterodimers involving the linkage of a BKAnpeptide and another peptide of different receptor activity through thelinking group X, e.g. an NK₁ or NK₂ antagonist peptide or a mu-opioidreceptor agonist peptide. Such heterodimers are particularly usefulwhere there is a close relationship between the activities of concern.Thus, it is known that in a number of pathophysiologically importantprocesses, there is an intimate interaction of inflammatory andneurogenic mediators. This occurs, for example, in both pain secondaryto tissue trauma (accidental and post-operative) as well as in asthma.In both situations, there is a complex interplay of tissue and plasmaderived mediators (such as kinins acting at BK₂ receptors) andneuronally derived factors such as substance P (NK₁ receptors) andneurokinin A (NK₂ receptors). There are also locally acting neuronalreceptors of the mu-opiate class that when stimulated can inhibit therelease of the neurogenic peptides regardless of type (substances P,neurokinin A, neurokinin B, cholecystokinin, CGRP, etc.).

Given the interaction of these as well as other inflammatory andneurogenic mediators, no one agent is likely to be universallyefficacious in ameliorating the symptoms attendant to thepathophysiology. The heterodimers described in the above-notedapplications are directed towards addressing these problems with singleagents possessing dual selectivity. Other advantages of suchheterodimers will also be appreciated by those in the art.

BRIEF DESCRIPTION OF THE INVENTION

The present invention, in its broadest aspect, is concerned withheterodimers obtained by linking a BKAn peptide to another pharmacophorewhich is not a bradykinin antagonist, i.e. which may be either a peptideas described in the afore-mentioned applications Ser. No. 07/677,381 andSer. No. 07/859,582, or a non-peptide, effective against a different,non-bradykinin component responsible, for example, for pain and/or theinflammatory process, or other problems related to or occurring inconcert with the activity of the kinins. The resulting compounds are"dual action" compounds that are capable of interacting with tworeceptor populations or, alternatively, with a receptor and an enzyme.This is not intended to suggest that the single molecule will engage tworeceptors or a receptor and an enzyme simultaneously; only that themolecule is capable of interacting with either one of two receptor typesor with a single class of receptors and/or an enzyme. The overallpharmacological effect of administering such a compound in anappropriate dose, however, is at least the summation of the two types ofactivities. The compounds can be designed to remain intact or they canbe designed to be dissociated into two separate molecules each retainingits own identifiable activity.

The heterodimers of the invention can be structurally represented asfollows:

    (Y)(X)(BKAn)

where BKAn is a bradykinin antagonist peptide; X is a linking group andY is a peptide or non-peptide pharmacophore which is not a bradykininantagonist and demonstrates activity towards a different receptor orenzyme than the BKAn component, preferably one related to pain or theinflammatory process.

The present heterodimers offer the possibility of providing a widerspectrum of treatment for pain and inflammation. It is a generally heldopinion that in inflammatory states, regardless of severity, thelikelihood that a single agent or mediator is completely responsible forall of the clinical manifestations of the syndrome being addressed isextraordinarily small. A corollary to this is that, given the role ofbradykinin in inflammatory pathophysiology, any combination therapy usedin the treatment of inflammatory disorders should include bradykininantagonism as part of its overall profile of action. Broad spectrum andpotent non-specific therapies (such as the use of steroids in asthma)while perhaps efficacious, carry with them the burdens of undesired andpotentially serious side effects and toxicities.

In many cases, two discrete mediators are known to act synergisticallyand to account for an overwhelming proportion of the clinicallyimportant manifestations of the disease being treated. Such is the case,for example, with substance-P acting at NK₁ receptors and bradykininacting at BK₂ receptors in the contexts of asthma and post-traumatic orpost-operative pain. Similarly, neutrophil elastase as one of the moreimportant down stream effectors of inflammation and bradykinin as one ofthe more important initiating and sustaining inflammatory mediators alsocan be viewed as being synergistic in their actions.

The concept of providing homodimers of pharmaceutically active materialsto improve such characteristics as metabolic stability, selectivity andreceptor binding has previously been described for other systems. Thisprior work has included the dimerization of peptide agonists andantagonists in order to increase potency and/or duration of action. See,Caporale et al, Proc. 10th American Peptide Symp., Pierce Chemical Co.,Rockford, Ill. 449-451 (1988) and Rosenblatt et al, European PatentApplication No. EP 293130A2. Thus, dimerization of peptide agonists hasbeen disclosed for enkephalins/endorphins (Shimohigashi, Y., et al,BBRC, 146, 1109-1115, 1987); substance P (Higuchi, Y., et al, E.J.P.,160, 413-416, 1989); bradykinin (Vavrek, R. and Stewart, J., J. Proc.8th Amer. Pept. Symp., 381-384, 1983); neurokinin A & B, (Kodama, H., etal, E.J.P., 151, 317-320, 1988); insulin (Roth, R. A., et al, FEBS, 170,360-364, 1984) and atrial natriuretic peptide (Chino, N., et al, BBRC,141, 665-672, 1986). Dimerization of antagonists has been shown forparathyroid hormone (Caproale, L. H., et al, Proc. 10th Amer. Pept.Symp., 449-451, 1987)). However, the literature has not disclosedheterodimers comprised of a bradykinin antagonist and a differentpharmacophore as contemplated herein.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A and 1B show structural details of some of the compounds or thepresent invention and some intermediates used in the preparationthereof.

FIGS. 2A and 2B show the results obtained with oxymorphone in the mouseformalin test.

FIGS. 3A and 3B show the results obtained with compound CP-0127 in themouse formalin test.

FIGS. 4A and 4B show the results obtained with compound CP-0494 in themouse formalin test.

FIGS. 5A and 5B show the results of reverse phase HPLC analysis ofmetabolites after incubating compound CP-0502 with human plasma,followed by acid precipitation of plasma proteins.

DETAILED DESCRIPTION OF THE INVENTION

Numerous bradykinin antagonist peptides are known in the art and any ofthese may be used for present purposes to provide the BKAn substituentof the present dimers. One of the more potent bradykinin antagonists invitro is the peptide having the formula:

    D-ARG.sup.0 -Arg.sup.1 -Pro.sup.2 -Hyp.sup.3 -Gly.sup.4 -Phe.sup.5 -Ser.sup.6 -D-Phe.sup.7 -Leu.sup.8 -Arg.sup.9

See Regoli et al, Trends in Pharmacological Science, 11:156-161 (1990).This peptide is referred to herein for convenience as CP-0088.

While CP-0088 is a convenient BKAn to use, those in the art willappreciate that other available or known bradykinin antagonist peptidescan also be used for present purposes. A wide variety of such bradykininantagonist peptides have been disclosed in the recent patent literatureand any of these can be used for present purposes. See, for example,EP-A-0334244 (Procter and Gamble) which discloses nona- and largerbradykinin antagonist peptides in which certain amino acid residues aremodified. EP-A-0370453 (Hoechst) and WO 89/01780 and WO 89/01781(Stewart et al) also describe bradykinin antagonist peptides. None ofthese patent publications appears to show dimers as contemplated herein.However, as noted, the peptides of these publications can be used in thepractice of the present invention.

Any linking group X may be used for present purposes to chemically orcovalently link together the BKAn and Y components provided this doesnot interfere with the activity of the components BKAn and Y. Thelinking group may be inorganic (e.g. --S--) or organic and may beselected so as to hydrolyze or otherwise dissociate in order to liberatethe two active components BKAn and Y in vivo. Alternatively, the linkinggroup may be such that the heterodimer remains intact when used.

Conveniently the linking group X can include an --S-- atom derived byreacting a sulfhydryl group on the BKAn peptide chain with the otherpharmacophore component. This can be accomplished by reaction involvinga cysteine (Cys) sulfhydryl group within the peptide chain, i.e.intermediate the ends of the peptide. This may require initiallymodifying the starting BKAn peptide so that it includes a Cys group inthe appropriate position in the peptide chain. For example, CP-0088 maybe modified by replacing the Ser in the 6-position with Cys (suchmodified CP-0088 being called CP-0126 hereinafter) to provide forconvenient linking to the other pharmacophore through the Cyssulfhydryl.

CP-0126 can be structurally illustrated as follows: ##STR1## Inabbreviated fashion, the formula may be stated as:

    DR-R-P-J-G-F-C-DF-L-R

Using Cys as the position of attachment, the linking group X thenincludes the --S-- of the cysteine sulfhydryl. This may be the entirelinking group X (as in a disulfide based dimer) or only a part thereof.Thus, for example, the linking group may comprise a bissuccinimidoalkanesuch as bissuccinimidohexane joined at its end to the BKAn and Ycomponents. These and other linking groups are disclosed in the relatedapplications referred to earlier herein and any of these may be used forpresent purposes. Other linking groups X, some of which do not requireor contained an --S-- atom, can be derived from the six families ofcompounds listed below which can be generically categorized as aminoacid analog linkers or maleimide-based linkers. These linkers areincluded as examples only and are not intended to be totally inclusiveof all potential linking moieties: ##STR2##

The amino acid analog linkers (Class I) can be directly incorporatedinto the peptide chain of the BKAn and then used to form esterase stableor labile heterodimers with the geminal pharmacophore (component Y).Alternatively, the maleimide based linker can be reacted with thedesired pharmacophore and then conjugated to a sulfhydryl containingpeptide. Finally, linkers from any of these families of compounds whichcontain --CO₂ H as the R₃ moiety can be reacted with another linker fromthese classes of compounds to form esterase labile (R₃ =--OH containinglinkers) or esterase stable (R₃ =--NH₂ containing linkers) which canthen be used to form the desired peptide/non-peptide heterodimer. R₁ andR₂ can be varied so as to provide for completely unhindered orsignificantly hindered access to the carbonyl carbon of an ester basedlinking element so that the rate of in vivo hydrolysis of said ester canbe controlled as desired.

Certain of the linker-modified BKAns or pharmacophores used herein toprepare the dimers of the invention are themselves novel and constitutea further aspect of the invention.

The component Y of the present heterodimers may be any peptide ornon-peptide pharmacophore, other than a bradykinin antagonist, whichdemonstrates activity towards a different (non-bradykinin) componentrelated to pain and/or the inflammatory process so as to provide dualaction compounds that are capable of interacting independently with twodifferent receptor populations or a receptor and an enzyme. Thus, forexample, the component Y may be a non-peptide mu-opioid receptoragonist, e.g. morphine or one of its derivatives such as oxycodone oroxymorphone.

Indomethacin is a useful choice for the Y component when cyclooxygenaseinhibition (COI) is desired. However, any of the other conventionalnon-steroidal anti-inflammatory agents, such as aspirin, ibuprofen,naproxyn or the like can be used. In this case, the BKAn/COI heterodimermay need to be hydrolyzed in order to obtain in vivo COI activity ascyclooxygenase is generally considered to be an intra-cellular enzyme.

Where neutrophil elastase inhibition is required, this may be an activeester, e.g. one of those described in U.S. applications Ser. No.07/528,967, filed May 22, 1990; Ser. No. 07/692,322, filed May 2, 1991or Ser. No. 07/841,608, filed Feb. 25, 1992, the contents of which areincorporated herein by reference. The esters described in saidapplications are 2-phenyl-alkanoate esters. There may also be used aheteroaryl alkanoate esterase inhibitor as described in Ser. No.07/866,301, filed Apr. 13, 1990 and Ser. No. 07/610,207, filed Nov. 7,1990, the contents of which are included herein by reference. Apreferred neutrophil elastase inhibitor for use herein as component Y isidentified below as CE-1218. This is believed to be a new compound andconstitutes a further feature of the present invention.

Other types of elastase inhibitors which may also be used as componentY, include fluoromethyl ketones, phosphonates, benzoxazoles,beta-lactams, etc.

As noted earlier, component Y may comprise a peptide or non-peptideinhibitor having a desired activity other than bradykinin antagonistactivity. However, the Y component is preferably selected to provideactivity against receptors or enzymes which have a common or closerelationship to the activity of bradykinin, e.g. the treatment of painor inflammation. The rationale for using combinations of a BKAn with amu-opioid receptor agonist, neutrophil elastase inhibitor,cyclooxygenase inhibitor or NK₁ or NK₂ receptor antagonist in variousconditions is discussed below for purposes of illustration.

BKAn/mu-opioid Receptor Agonists

C-Fiber afferents are known to mediate both the sensation of pain aswell as the neurogenic component of inflammation. These afferent neuronsrelease a variety of neuropeptides in response to specific andnon-specific stimuli in both the central nervous system (CNS) as well asin the peripherally innervated tissues. Some of these neuropeptidesinclude: substance-P, neurokinin A, neurokinin B, calcitonin generelated peptide (CGRP), cholecystokinin (CCK), vasoactive intestinalpolypeptide (VIP), and neuropeptide Y, among other neurotransmitters. Toadd to this complexity, different C-fibers appear to contain differentamounts and/or ratios of these neuropeptides depending on the tissueinnervated. All of these peptides have been shown to play contributoryroles in the various neurogenic processes that have been implicated innumerous diseases and clinical syndromes. In fact, specific antagoniststo these peptides are being developed as potential therapeutics by avariety of pharmaceutical companies and independent researchlaboratories.

One apparently common feature among this otherwise diverse group ofneurons is that they all have mu-opioid receptors that modulate therelease of these neuropeptides. Both the endogenous enkephalins as wellas other exogenously administered small molecular weight compounds suchas morphine, oxymorphone, fentanyl and their derivatives will inhibitthe release of the neuropeptides from peripheral C-fibers by acting asmu-opioid receptor agonists locally (at terminal mu-opioid receptors inthe periphery) and in the CNS. This inhibition is independent of boththe constellation of peptides contained in the specific C-fiber as wellas the stimulus causing their release.

As a result, one important class of compounds considered to have aparticularly good profile of activities for the treatment of conditionsthat are produced by combined humoral and neurogenic processes areBKAn/mu-opioid receptor agonist heterodimers. These compounds would beexpected to attenuate or block both the humoral component of theinflammatory process as represented by the kinins as well as theneurogenic aspects of inflammation produced by the release of theneuropeptides. In addition, one of the limiting aspects of the use ofexisting mu opioid agonists is their propensity to produce sedation,confusion, and a depressed respiratory drive, not to mention theirpotential for the development of addiction and/or tolerance in thepatients being treated with these agents. These undesirable aspects ofmu-opioid receptor agonists are due to their ability to easily penetratethe CNS. BKAn/mu-opoid receptor agonist heterodimers, however, shouldnot penetrate the CNS due to the highly cationic nature of the BKAn.Consequently, mu-opoid receptor agonist activity should be limited tothe periphery and should result in a substantially reduced sideeffect/toxicity profile for these types of compounds.

BKAn/Neutrophil Elastase Inhibitor (NEI)

As previously mentioned the control of both systemic and localinflammatory responses may require interventions in more than oneinflammatory pathway. In particular, the ability to block the activityof a primary mediator responsible for the initiation and maintenance ofthe inflammatory process (such as bradykinin) and a primary finalpathway effector responsible for actual tissue degradation and injury(such as neutrophil elastase) may be a key to "single drug therapy" ofsepsis or other severe inflammatory conditions requiring parenteraltherapy or for the treatment of inflammatory dermatologic ordental/periodontal conditions.

Heterodimers containing combined BKAn/NEI activities can be designed toremain intact or to be dissociable as both targets (bradykinin receptorsand neutrophil elastase) are extracellular in nature. However, shoulddissociation of the two active pharmacophores be desired, linkingmoieties tethering the two active components of the heterodimer can bedesigned to be hydrolyzed by, for example, plasma hydrolases. Thesetypes of dissociable or hydrolyzable heterodimers are discussed herein.

BKAn/Cyclooxygenase Inhibitor (COI)

A large proportion of the biological activity of bradykinin isinterwoven with the generation of prostaglandins. For example, much ofthe hyperalgesia associated with inflammatory pain appears to bedependent on the generation of certain prostaglandins both by theinjured tissues and by the C-fibers themselves. In the latter case,bradykinin and substance-P appear to be the primary stimuli for these"second messengers". The local generation of prostaglandins by theinjured tissues is bradykinin independent. This interaction of peptidepro-inflammatory mediators and prostaglandins occurs in other settingsas well and can also be considered a target for dual action compounds.Heterodimers containing combined BKAn/COI activities may need to undergoin vivo dissociation of the respective pharmacophores as cyclooxygenaseis an intracellular enzyme and functional bradykinin receptors arelimited to the external plasma membrane.

BKAn/NK₁ -Receptor Antagonist (NKlAn)

Bradykinin and substance-P are known to act synergistically in theinitiation and maintenance of the inflammatory and neurogenic componentsof both asthma and a variety of painful conditions. In both of thesesituations, bradykinin is one of the more potent, if not the mostpotent, agents capable of stimulating C-fiber sensory afferents thatmediate peripheral pain and/or the sensation of cough and dyspnea inasthma. These neurons, regardless of the primary stimulus, will releasesubstance-P which amplifies and augments the activity of bradykinin andother stimuli at the sensory nerve endings where these stimuli areacting. This "one/two punch" of initial stimulus followed by localamplification is well documented and has significant implications forthe success or failure of any single intervention. By targeting bothcomponents of these processes with a single compound, it is possible toprovide a dually-specific agent which is superior than mono-specificagents used alone and both easier and cheaper to use than combinationtherapies.

BKAn/NK₂ Antagonist (NK2An)

Bradykinin's ability to produce acute bronchial smooth muscleconstriction is at least partially dependent on the release ofneurokinin A by the same C-fibers that release substance-P. Neurokinin Aexerts its effect via NK₂ receptors on the bronchial smooth muscle.However, more than just bradykinin can release neurokinin A from theseneurons and, as a result, a dually-specific antagonist with combined BK₂/NK₂ antagonist activity should provide better overall amelioration ofbronchoconstriction in the asthmatic patient than any other singleagent.

The heterodimers of the invention may be prepared in generally the samemanner as described in the above-mentioned Ser. No. 07/859,582 and Ser.No. 07/677,391. Normally this involves adding the linking group X to theBKAn component at an appropriate position along the peptide chainfollowed by joining the non-peptide pharmacophore to the BKAn throughthe linking group. Alternatively, the linking group may be added to thenon-peptide pharmacophore and the BKAn thereafter joined to thelinker-modified pharmacophore. Representative procedures are describedbelow although it will be recognized that various modifications may beused.

The invention is illustrated but not limited by the following examples:

EXAMPLES 1-4 (BKAN/MU-OPIOID AGONIST)

Four different peptide/opiate heterodimers (designated CP-0477, CP-0488,CP-0494 and CP-0499) were made in order to illustrate the invention.Three of these compounds were made using CP-0126 (DR-R-P-J-G-F-C-DF-L-R)and the fourth used CP-0347 (DR-R-P-J-G-Thi-C-DTic-Oic-R). Similarly,two different opiates (oxycodone and oxymorphone) and two differentlinker chemistries were used to provide the respective heterodimers asfollows:

    ______________________________________                                        Example  Compound #   Peptide  Opiate                                         ______________________________________                                        1        CP-0477      CP-0126  Oxycodone                                        2 CP-0488 CP-0126 Oxycodone                                                   3 CP-0494 CP-0126 Oxymorphone                                                 4 CP-0499 CP-0347 Oxymorphone                                               ______________________________________                                    

The heterodimers CP-0477, CP-0488, CP-0494 and CP-0499 were prepared asdetailed hereinafter with reference to the accompanying FIG. 1:

Preparation of Compound I

Oxycodone hydrochloride (0.182 g, 0.52 mmol), acetic acid (0.475 ml, 8.3mmol), S-benzyl cysteamine (0.174 g, 1.04 mmol) and methanol (5 ml) werecombined and stirred at room temperature for an hour. Sodiumcyanoborohydride (95%, 0.033 g, 0.52 mmol) was added, and the reactionstirred at room temperature for 24 h. The mixture was concentrated invacuo. The resulting oil was dissolved in ethyl acetate and the ethylacetate fraction was washed with saturated sodium bicarbonate solution,dried over magnesium sulfate and evaporated in vacuo. The crude materialwas chromatographed on a silica column and eluted with EtOAc, EtOAc-MeOH(9:1, v/v) and EtOAcMeOH-Et₃ N (9:1:0.2, v/v/v) successively Compound Iwas isolated as an oil in 25.0% (59.0 mg) yield.

Preparation of Compound II (CP-0477)

I (0.059 g, 0.127 mmol) was dissolved in 2 mL dry tetrahydrofuran, andwas transferred to a oven dried three-necked 100 ml flask. The flask wasfitted with a dewar condenser, a nitrogen source and an ammonia inlet.Approximately 10 ml of ammonia was condensed into the flask maintainedat -78 C. Small pieces of sodium were added until the intense blue colorwas maintained and then quenched after 40 seconds with solid ammoniumchloride. The reaction mixture was allowed to warm to room temperatureand the ammonia boiled off through a bubbler, methanol (25 ml×3) wasadded and evaporated in vacuo. The thiol isolated was dissolved in aminimum quantity of DMF (N, N-dimethyl formamide, 2 ml). Compound X(approximately 0.3 equiv) was dissolved in tris buffer (0.5 M, 4.0 ml)and added to the DMF solution and then stirred for 17 h. The crudemixture was purified on a reverse phase Vydac C-18 HPLC column using thegradient 15-40% CH₃ CN in water, 0.1% constant TFA, over 20 minutes.Retention time was 16.0 minutes. 26.4 mg of II was isolated as a whitepowder on lyophillization.

Analysis

The mass spectra was run on a Finnigan Lasermat Mass Analyzer.

calulated molecular weight--1916

observed molecular weight--1918

Amino Acid Analysis

Gly 1.02 (1), Arg 3.14 (3), Pro 1.01 (1), Leu 0.97 (1), Phe 1.92 (2) andHyp 0.94 (1).

Preparation of Compound III

To the mixture of oxycodone hydrochloride (1.0 g, 2.84 mmol) andammonium acetate (2.2 g, 28.4 mmol) dissolved in methanol (10.0 ml) wasadded a methanolic (4.0 ml) solution of NaCNBH₃ (0 18 g, 2.84 mmol). Theresulting solution was adjusted to pH 7.0 with concentrated hydrochloricacid, stirred for 17 h, and acidified to pH 1.0 with concentratedhydrochloric acid. The solvent was removed in vacuo and the remainingmaterial was dissolved in water. The aqueous layer was extracted withchloroform, adjusted to pH 9.0 with 10% sodium carbonate solution,saturated with NaCl and extracted with chloroform. The chloroform layerwas dried over magnesium sulfate and evaporated in vacuo. The crude oilwas purified by silica gel chromatography and eluted with EtOAc,EtOAc-MeOH (9:1, v/v), EtOAc-MeOH-Et₃ N (9:1:0.3, v/v/v) successively.Compound III was isolated as an oil in 47 0% (0.42 g) yield.

Preparation of Compound IV

BOC-Glycine (0.16 g, 0.91 mmol), HOBt (0.125 g, 0.91 mmol) and1-(3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (980%, 0.18 g, 0.91 mmol) were dissolved in DMF (2.0 ml) and stirred at 0°C. for an hour. The amine III (0.24 g, 0.76 mmol) dissolved in DMF (3.0ml) was added to the reaction mixture, the reaction mixture was warmedto room temperature and stirred for 17 h. DMF was removed in vacuo andthe resulting material was dissolved in ethyl acetate. The ethyl acetatelayer was washed with saturated sodium bicarbonate solution, brine anddried over magnesium sulfate. The organic layer was evaporated in vacuoand the crude mixture was flash chromatograhed on a silica gel columnand eluted with EtOAc-MeOH-Et₃ N (9.5:0.5:0.3, v/v/v). Compound IV wasisolated as an oil in 82.0% (0.29 g) yield.

Preparation of Compound V

The BOC protecting group was removed off the compound IV with TFA (5.0ml) in methylene chloride (5.0 ml). Methylene chloride was removed invacuo and the residue was stripped with methylene chloride (20 ml×3) andthen with triethyl amine (3 ml×3). 3-S-benzyl mercapto propionic acid(0.15 g, 0.75 mmol), EDC (0.15 g, 0.75 mmol), HOBt (0.103 g, 0.75 mmol)and Et3N (0.35 ml, 2.48 mmol)were dissolved in DMF (5.0 ml) and stirredat 0° C. for an hour. The solution of the amine (0.23 g, 0.62 mmol) inDMF (3.0 ml) was added to the reaction mixture. The reaction mixture waswarmed to room temperature and stirred for 17 h. DMF was evaporated invacuo and the residue was dissolved in EtOAc. The EtOAc layer was washedwith 10% Na₂ CO₃, brine, dried (over MgSO₄) and evaporated in vacuo. Thecrude material was purified on a flash silica gel column and eluted withEtOAc-MeOH-Et₃ N (9:1:0.3, v/v/v). Compound V was isolated as an oil in60.0% (0.205 g) yield.

Preparation of Compound VI (CP-0488)

32.0 mg (0.057 mmol) of V was deprotected using the procedure describedfor II and the thiol isolated was reacted with compound X (0.073 g,0.048 mmol) in tris buffer. The crude mixture was purified using theprocedure for II. Retention time of the product was 16.82 minutes. 9.5mg (10%) of VI was obtained as a white powder on lyophillization.

Mass Spectral Data

calulated molecular weight 2,002

observed molecular weight 2,004

Amino Acid Analysis

Gly 1.76 (2), Arg 3.19 (3), Pro 1.06 (1), Leu 0.99 (1), Phe 2.06 (2),Hyp 0.95 (1).

Preparation of Compound VII

Oxymorphone hydrochloride (0.56 g, 1.66 mmol), S-benzyl cysteamine (0.69g, 4.15 mmol), acetic acid (1.52 ml, 26.5 mmol) and sodiumcyanoborohydride (0.11 g, 1.66 mmol) were used according to theprocedure for I. The composition of the third eluant, EtOAc-MeOH-Et₃ N,used in the purification of the crude mixture was 9:1:0.3, v/v/v. Onpurification, 0.213 g of compound VII (29.0%) was isolated as an oil.

Preparation of Compound VIII (CP-0494)

VII (0.063 g, 0.14 mmol) was deprotected following the procedure for II.The thiol was then treated with X (0.335 g, 0.152 mmol) in tris buffer.The crude mixture was purified on a reverse phase Vydac C-18 HPLC columnusing the gradient 15-70% CH₃ CN in water, 0.1% constant TFA over 35minutes. VIII had a retention time of 15.0 minutes. 119.0 mg (45.0%) ofVII was isolated as a white powder on lyophillization.

Mass Spectral Data

calculated molecular weight 1902

observed molecular weight 1904

Amino Acid Analysis

Gly 0.81 (1), Arg 3.12 (3), Pro 1.07 (1), Leu 0.99 (1), Phe 2.04 (2),Hyp 0.98 (1).

Preparation of Compound IX (CP-0499)

VII (0.009 g, 0.02 mmol) was deprotected following the procedure for IIand the thiol was then reacted with XI (0.026 g, 0.016 mmol) in trisbuffer. Crude mixture was purified on a Vydac C-18 reverse phase HPLCusing the gradient 15-70% CH₃ CN in water, 0.1% constant TFA, flow rateof 8.0 ml/min over 40 minutes. Retention time of IX was 14.22 minutes.IX (6.4 mg, 20.0%) was isolated as white powder on lyophillization.

Mass Spectral Data

Calculatedmolecularweight 1957

Observed molecular weight 1958

IN VITRO TESTING

The BKAn/mu-opioid receptor agonist heterodimers were evaluated in vitrousing the rat uterus (BK₂ -receptor activity) and the electricallystimulated guinea pig ileum (mu-opiate receptor activity) assays. Theseassays are well known in the art. The results obtained are shown inTable I:

                  TABLE I                                                         ______________________________________                                                                IC.sub.50 Guinea Pig Ileum                              Compound pA.sub.2 --Rat Uterus (nmolar)                                     ______________________________________                                        CP-0126     7.1         inactive                                                CP-0347 9.5 inactive                                                          oxycodone inactive inactive                                                   oxymorphone inactive 21.7                                                     CP-0477 7.9 inactive                                                          CP-O488 8.2 inactive                                                          CP-O494 8.4 24.0                                                              CP-0499 8.9 17.0                                                            ______________________________________                                    

It should be noted that neither oxycodone nor the heterodimers derivedfrom oxycodone (CP-0477 and CP-0488) showed any activity in the in vitroguinea pig ileum assay of mu-opiate receptor agonist activity. This isprobably due to the fact that for complete activity, oxycodoneapparently needs to be demethylated in vivo. As a result, oxycodone andoxycodone-based compounds would not be expected to show activity in anassay wherein the appropriate demethylating enzymes were missing.

More important, however, are the data regarding the activity of the BKAncomponent of these heterodimers on the rat uterus and the data regardingthe activity of the oxymorphone containing compounds. As can be seenfrom the data outlined in Table I, full BKAn activity was retained inall of these heterodimers and in those compounds utilizing oxymorphoneas the opiate, full mu-opiate receptor agonist activity was alsoretained. From these data, it is evident that BKAn/mu-opioid receptoragonist heterodimers can interact with their respective receptorpopulations in in vitro systems.

IN VIVO TESTING

In order to test the activity of these compounds in vivo, a model ofinflammatory and neurogenic pain was used. This model measures thebehavioral responses of mice injected in the hind limb foot pad with 50ul of formalin. The data from these studies are summarized in FIGS. 2, 3and 4. Control mice (open circles) show a characteristic bi-phasicresponse to the injected formalin wherein there is a short lastinginitial response followed by a quiescent period which is then followedby a sustained period of hind limb licking. The licking behavior isinterpreted to mean that the limb is irritated and painful. The greaterthe time spent licking, the more painful the stimulus.

Oxymorphone (FIG. 2A and B) reduces both phases of the licking behaviorbut with significant behavioral obtundation resulting in catalepsy andfrank respiratory depression at the highest doses (0.9 and 3.0umoles/kg). The bradykinin antagonist CP-0127 (a potent BK₂ selectiveantagonist--FIGS. 3A and B) will reduce the time spent licking in bothphases of the formalin test but at doses that are substantially greaterthan would be practical in a clinical setting. CP-0494 (FIGS. 4A and B),however, not only blocks both phases of the pain response, but does soat doses substantially lower (0.1 umoles/kg) than for either oxymorphone(0.9 umoles/kg) or CP-0127 (12.6 umoles/kg) alone and, of equal orgreater importance, with no observable narcotic effects over severalhours. These data indicate that BKAn/mu-opioid receptor agonistheterodimers are pharmacologically qualitatively superior to either ofthe parent pharmacophores as would be expected from the theoreticalconsiderations outlined above.

One skilled in the art will appreciate that the compounds described arerepresentative of a wide variety of compounds in which each of thecomponents of the heterodimer (BKAn, linker and/or mu-opioid receptoragonist) can be varied to produce the optimal effect desired.

EXAMPLE 5 (BKAn/NEI)

A BKAn/NEI type of compound (CP-0502) of the structure shown inSynthetic Scheme 3, was synthesized to illustrate that this class ofcompounds can be used as a potent topical and/or systemicanti-inflammatory agent. This compound is derived from CP-0126 and theprototype elastase inhibitor, CE-1218 (see Compound (6), SyntheticScheme 1 below). ##STR3##

The linking element used in this heterodimer was chose so as to allowfor unhindered hydrolysis of the joining ester bond by serum esterases.Those skilled in the art will recognize that the linker can be modifiedso as to provide different rates of hydrolysis varying from rapid topractically zero by altering the steric accessibility of the estercarbonyl carbon or by changing the chemistry to an amide linkage.Completely stable linker moieties can also be used which are free frompotential hydrolytic degradation.

Synthesis and Analysis of BKAn/NEI Heterodimers

The synthesis of these compounds is illustrated by reference toSynthetic Schemes 2 and 3 and the following detailed synthesisdescription which includes the preparation of the elastase inhibitorCE-1218 according to Synthetic Scheme 1:

SYNTHETIC SCHEME 1

Synthesis of 4-tert-Butylacetophenone (1)

To a dry 1-L flask was added CS₂ (250 mL) and AlCl₃, (133.34 g, 0.56mol) with stirring. The suspension was cooled in an ice bath and asolution of tert-butylbenzene (50.00 g, 0.37 mol) and acetyl chloride(78.50 g, 0.41 mol) was added dropwise over 2 hr (not allowing thetemperature to rise above 25° C.). The reaction was allowed to stir atroom temperature overnight and then poured into a 2 L beaker filled withice. After quenching with 200 mL of 6 N HCl the solution was saturatedwith NaCl and separated. The aqueous layer was washed with ether (2×100mL) and combined with previous organics. This new organic solution waswashed with water (100 mL), dried (MgSo₄) and evaporated to give an oilwhich was distilled to give 52.1 g (79.3%) of 4-tert-acetophenone as aclear colorless oil (bp₀.05 mm 70-76° C.). ¹ H NMR (CDCl₃) δ 1.35 (s, 9H), 2.58(s, 3 H), 7.48 (d, J=8.5 Hz, 2 H), 7.91 (d, J=8.5 Hz, 2 H). ¹³ CNMR (CDCl₃) δ 26.42, 30.96, 34.95, 125.36, 128.16, 134.49, 156.64,197.61.

Synthesis of Methyl 4-tert-butylphenylacetate (2)

A dry 1 L flask equipped with a mechanical stirrer containing Pb(OAc)₄(132.06 g, 0.298 mol) and 250 mL of benzene was purged with nitrogen andcooled in an ice bath. To this cooled slurry was added dropwise asolution of BF₃ OEt₂ (137.8 mL, 1.12 mol), 4-tert-butylacetophenone(50.00 g, 0.284 mol) in 70 mL of methanol over 1 hr. This mixture wasallowed to stir overnight, quench with water (500 mL), diluted with 250mL ether and the layers separated. The organic layer was washed withwater, diluted NaHCO₃ (carefully) and dried over MgSO₄. The mixture wasfiltered, evaporated and distilled to give 31.2 g (53.4%) of methyl4-tert-butylphenylacetate as clear colorless oil (bp₀.04 mm 75-80° C.).¹ H NMR (CDCl₃) 1.32 (s, 9 H), 3.62 (s, 2 H), 3.71 (s, 3 H), 7.23 (d,J=8.4 Hz, 2 H), 7.37 (d, J=8.4 Hz, 2 H). ¹³ C (CDCl₃) 31.33, 34.46,40.67, 52.04, 125.53, 128.88, 130.91, 149.94, 172.26.

Synthesis of Methyl 4-tert-butylphenylisobutyrate (3)

A solution of methyl 4-tert-butylphenylacetate (30.00 g, 0.145 mol) andiodomethane (45.41 g, 0.320 mol) in 125 mL of dry THF was added to aslurry of NaH (8.72 g, 0.363 mol) in 200 mL of THF dropwise over 30minutes. After completion of the addition, the reaction mixture washeated at reflux for 1.5 hr. The reaction was allowed to cool to roomtemperature, filtered through Celite and concentrated. The residue wasdiluted in ether, washed with H₂ O, and dried over MgSO₄. Evaporation ofthe solvent afforded the desired product as an oil. A mixture of thecrude methyl 4-tert-butylphenylisobutyrate and 4:1 EtOH/H₂ O containingKOH (10.07 g, 0.179 mol) were heated to reflux for 4 hr. The EtOH wasevaporated in vacuo, the residual solution was acidified to pH 2 with 2N HCl, and the precipitated solid filtered. The white solid was thedried (60° C., 1 mm Hg, 24 hr) to give the desired product (23.45 g,73.2% from methyl 4-tert-butylphenylacetate). ¹ H NMR (CDCl₃) 1.34 (s, 9H), 1.62 (s, 6 H), 7.37 (s, 4 H), 11.4-12.4 (brs, 1 H). ¹³ C NMR (CDCl₃)26.16, 31.30, 34.35, 45.81, 125.31, 125.48, 140.64, 149.66, 183.57.

Synthesis of 4-(3'-carbo-tert-butoxy-propyl mercapto)phenyl4-tert-butylphenylisobutyrate (4)

A mixture of 4-tert-butylphenylisobutyric acid (2.00 g, 0.0091 mol) andthionyl chloride (1.62 g, 0.0136 mol) in 16 ml of CH₂ Cl₂ was allowed tostir overnight under Argon. The volatiles were removed under vacuum andthe resulting solid was dissolved into THF (15 mL) and a solution oftert-butyl-4-(4'-hydroxyphenyl)mercaptobutyrate (2.44 g, 0.0091 mol),TEA (2.5 mL) in THF (15 mL) was added dropwise over 10 min. The mixturewas stirred for 3 days, diluted with Et₂ O and extracted with 5% NaHCO₃.The organics were washed with H₂ O, brine and dried (MgSO₄). Afterevaporization the colored oil was separated (HPLC, silica gel 70:30 CH₂Cl₂ /hexane to CH₂ Cl₂ linear graduent) to give the desired product asan oil (2.20 g, 51.5%). ¹ H NMR (CDCl₃) δ 1.33 (s, 9 H), 1.43 (s, 9 H),1.70 (s, 6 H), 1.88 (tt, J=7.2 Hz, 2 H), 2.35 (t, J=7.2 Hz, 2 H), 2.90(t, J=7.2, 2 H), 6.92 (d, J=8.6 Hz, 2 H), 7.32 (d, J=8.6 Hz, 2 H),7.34-7.40(m, 4 H). ¹³ C NMR (CDCl₃) δ 24.50, 26.42, 28.08, 31.31, 33.70,34.11, 34.39, 46.40, 80.40, 121.92, 125.23, 125.46, 130.82, 133.03,140.86, 149.58, 149.69, 172.21, 175.34.

Synthesis of 4-(3'-carboxy-propylmercapto)phenyl4-tert-butylphenylisobutyrate (5)

Trifluoroacetic acid (25 mL) was added to a stirred solution of4-(3'-carbo-tert-butoxy-propylmercapto)phenyl4-tert-butylphenylisobutrate (2.40 g, 0.00510 mol) in 20 mL of CH₂ Cl₂over 15 min. After an additional 15 min the volatiles were removed andthe oil crystallized (hexane) to give 1.94 g (91.8%) of desired productas a white solid, m.p. 86.0-87.0° C., ¹ H (CDCl₃) 1.33 (s, 9 H), 1.70(s, 6 H), 1.92 (tt, J=7.0 Hz, 2 H), 2.50 (t, J=7.0 Hz, 2 H), 2.93 (t,J=7.0 Hz, 2 H), 6.93 (d, J=8.7 Hz, 2 H), 7.33 (d, J=8.7 Hz, 2 H),7.35-7.39 (m, 4 H). ¹³ C NMR (CDCl₃) 23.89, 26.45, 31.32, 32.34, 33.63,34.42, 46.42, 122.03, 125.24, 125.48, 131.12, 132.63, 140.85, 149.74,175.40, 178.65.

Synthesis of 4-(3'-carboxy-propylsulfonyl)phenyl4-tert-butylphenylisobutyrate (6)

To a 50 mL flask was added 4-(3'-carboxy-propylsulfonyl)phenyl4-tert-butylphenylisobutyrate (1.64 g, 0.00396 mol), HOAc (25 mL) and 15mL of 30% H₂ O₂. The reaction was allowed to stir overnight, dilutedwith H₂ O (50 mL) and the resulting solid filtered. After drying (12 hr,1 mmHg) the solid was recrystallized (CH₂ Cl₂ /hexane) to give 1.54 g(87.1%) of the desired product as a white powder. mp 107-108.5° C. ¹ H(CDCl₃) 1.33 (s, 9 H), 172. (s, 6 H), 2.02 (p, J=7.0 Hz, 2 H), 2.52 (t,J=7.0 Hz, 2 H), 3.17 (t, J=7.0 Hz, 2 H), 7.20 (d, J=8.7 Hz, 2 H), 7.36(d, J=8.7 Hz, 2 H), 7.41 (d, J=8.6 Hz, 2 H), 7.90 (d, J=8.6 Hz, 2 H). ¹³C NMR (CDCl₃) 17.88, 26.29, 31.28, 31.79, 34.42, 46.53, 55.04, 122.56,125.16, 125.61, 129.70, 135.81, 140.22, 150.02, 155.29, 174.73, 177.71.

Synthesis of 6-Maleimidohexanol (7)

The synthesis of 6-maleimidohexanol to be used for linking isillustrated in Synthetic Scheme 2 below: ##STR4##

To a 100 mL flask was added 6-aminohexanol (0.76 g, 0.0064 mol) and 25mL of saturated NaHCO₃. The homogenous was allowed to stir a RT andN-methoxycarbonylmaleimide (1.00 g, 0.0064 mol) was added as a solid.The mixture cleared shortly after the addition and was allowed to stirfor 1 hr. The mixture was extracted by EtOAc, dried (MgSO₄) andevaporated. The resulting mixture was separated on silica gel (CH₂ Cl₂to EtOAC). To give the product as a white solid 0.32 g (25.2%), usedwithout further purification. ¹ H (CDCl₃) δ 1.25-1.45 (m, 4 H),1.45-1.70 (m, 4 H), 3.53 (t, J=7.3 Hz, 2 H), 3.63 (t, J=6.0 Hz), 6.71(s, 2 H).

Synthesis of CP-0502

Compound (6) (CE-1218) was esterified with compound (7) to form compound(8) which was then conjugated to CP-0126 to form the dimer CP-0502.These latter reactions are illustrated in Synthetic Scheme 3: ##STR5##

Referring more specifically to Synthetic Scheme 3, compound (6) (200 mg,0.448 mmol), triethylamine (0.124 ml, 2 eqv), 6-maleimidohexanol (7) (97mg. 1.1 eqv) were dissolved in 2 ml methylene chloride.Bis(2-oxo-3-oxazolidinyl) phosphinic chloride (122 mg, 1.0 eqv) wasadded to the stirred solution. The resulting suspension was stirred atroom temperature for four hours. The reaction mixture was diluted with25 ml methylene chloride and washed with saturated NaHCO₃. The organicsolution was dried over anhydrous MgSO₄ and the solvent was removed invacuo. Silica gel chromatography (2×18 cm column); 35/65:acetone/hexane(Rf=0.4) as eluent provided the compound (8) as a colorless oil.

Compound (8) (50 mg, 0.08 mmol) was dissolved in 10 ml DMF containing100 ul diisopropylethylamine. 100 mg (0.08 mmols) of CP-0126 was addedand reaction proceeded for 30 minutes, with occasional mixing. Thereaction mixture was injected on a Vydac 1" C-18 reverse phase column,and eluted at 10 ml/min, 15%-90% acetonitrile in H2O over 35 minutes(Constant 0.1% TFA). The appropriate fractions were lyophilized to yield52 mg (35%) of a colorless white powder (CP-0502) Laser desorption massspectrometry M/Z=1890 (M+H), calculated 1890. Automated amino acidsequence results confirmed the correct peptide sequence with no alteredamino acids.

In Vitro Activity of BKAn/NEI Heterodimer(s)

In vitro evaluation of BKAn and NEI activity of the following compoundswas carried out according to standard protocols well known to those inthe art. BKAn activity (pA₂) was assessed using the rat uteruspreparation and NEI (K_(i) ^(ss)) activity was evaluated using purifiedhuman neutrophil elastase (HNE) and a synthetic soluble chromogenicsubstrate, methoxysuccinyl-alanyl-alanyl-prolyl-valyl-paranitroanilinewith (MOS-AAPV-pNA). The inhibitor was mixed with MOS-AAPV-pNA (0.5 mM)in 0.05 M sodium phosphate, 0.1 M NaCl, 0.005% Triton X-100, 5% DMSO, pH7.5. HNE (10-20 nM) is then added. The production of nitroaniline wasmonitored spectrophotometrically at a wavelength of 400-410 nm at 25 C.An ENZFITTER program then automatically calculated standard enzymekinetic parameters including K_(i) ^(ss).

The following results were obtained:

                  TABLE II                                                        ______________________________________                                                                  K.sub.i.sup.ss (HNE)                                  Compound pA.sub.2 --Rat Uterus nM                                           ______________________________________                                        CP-0126       7.1         inactive                                              CE-1218 inactive 10.5                                                         CP-0487 8.4 inactive                                                          CP-0502 7.5  6.6                                                            ______________________________________                                    

The data in Table II indicate that for NEI activity there is littledifference between the intact heterodimer (CP-0502) and the freemonomeric NEI moiety (CE-1218) as far as their respective K_(i) 's areconcerned. This is not true for the activity of the BKAn portion of theintact heterodimer relative to its hydrolysis product, CP-0487 (thesuccinimidohexanol derivative of CP-0126) wherein the intact compound isalmost a full log less potent than the monomeric BKAn. Interestingly,the activity of the intact compound displayed a type of irreversiblebradykinin antagonism and an apparently enhanced antagonist activity ofbradykinin induced uterine contractions at longer incubation times.These types of receptor interactions are not well measured by standardpA₂ analyses so the differences in activity observed between CP-0487 andCP-0502 with respect to BKAn activity may be more apparent than real.Regardless of the molecular pharmacologic mechanisms underlying thesedata it is clear that combined BKAn and NEI activity can be incorporatedinto a single molecule.

The above data suggest that allowing for in vivo hydrolysis of theintact compound may alter the behavior of the two moieties so as toenhance the overall in vivo activity of the primary compound.Unfortunately, there are no established animal models that can beemployed to assess combined BKAn and NEI activity in vivo. Therefore, inorder to assess the potential for in vivo hydrolysis of the intactheterodimer, an in vitro "surrogate" system was employed wherein theparent heterodimer (CP-0502) was incubated with human plasma and theresulting metabolites analyzed by reverse phase HPLC.

CP-0502 was added to freshly obtained normal human plasma and allowed toincubate at 37° C. for varying amounts of time. At the designated timethe samples were treated with acidified (0.1 N HCl) acetonitrile inorder to precipitate the plasma proteins. Aliquots (75 ul) of thesupernatents were then analyzed on a Vydac C-18 reverse phase HPLCcolumn using 24% to 80% acetonitrile gradient in 0.1% TFA. The eluentwas monitored at 214 nm.

FIGS. 5a and b are representative reverse phase HPLC chromatogramsillustrative of this type of analysis. As can be seen from thesechromatograms, the parent compound appears to be readily hydrolyzed tothe succinimidohexanol modified monomer, CP-0487 and its des-Arg⁹derivative (Plasma carboxypeptidase will cleave the terminal arginineresidue from both the intact heterodimer, CP-0502, as well as CP-0487.)The apparent T_(1/2) of this hydrolysis reaction is approximately 113minutes. The NEI component of the heterodimer is an active ester andundergoes hydrolysis as well. However, the intact NEI monomer as well asits hydrolysis products are obscured by the plasma derived peaks seen inthe middle of this tracing and cannot be visualized using this system.Since the NEI is equally active as a component of the heterodimer as itis as a monomer, the dissociation of the heterodimer into its twocomponent parts will have less of an effect on its activity than thatfor the BKAn component.

Those skilled in the art will appreciate that the hydrolysis rate of theheterodimer can be influenced by the steric and electronic environmentof the "linking" ester moiety and that the type of chemistry used isonly a single example of the types of chemistry that can be employed toadjust the rate of dissociation (or lack thereof) of the two componentsof the heterodimer.

EXAMPLE 6 (BKAn/COI) Synthesis and Analysis of BKAn/COI Heterodimers

A representative BKAn/COI heterodimer (CP-0460) was synthesizedaccording to Synthetic Schemes 4 and 5 below: ##STR6## Synthesis of6-Maleimidohexanyl1-(4-chlorobenzoyl)-5-methoxy-2-methyl-3-indohylacetate (9)

To a 100 mL flask was added indomethacin (1.90 g, 0.00532 mol), 25 mL ofCH₂ Cl₂ and DCC (0.55 g, 0.00266 mol). After 2 hr, the mixture filtered,the DCU washed with 15 mL of CH₂ Cl₂ and to this new solution was added6-maleimidohexanol (0.50 g, 0.00253 mol) as a solid followed byanhydrous Na₂ CO₃ (0.32 g, 0.00304 mol). After 4 days the mixture wasfiltered diluted with Et₂ O and washed with 5% NaHCO₃, H₂ O and dried(MgSO₄). The resulting yellow oil was purified in a HPLC (silica gel;CH₂ Cl₂ to 80:20 CH₂ Cl₂ /EtOAc, linear gradient 60 min.) to give 0.79 g(58.0%) of the desired product as a yellow oil. ¹ H (CDCl₃) 1.20-1.35(m, 4 H), 2.38 (s, 3 H), 3.47 (t, J=7.3 Hz, 2 H), 3.66 (s, 2 H), 3.83(s, 3 H), 4.08 (t, J=6.6 Hz, 2 H), 6.66 (J=9.0 Hz, J=2.5 Hz, 1 H), 6.68(s, 1 H), 6.87 (d, J=9.0 Hz, 1 H), 6.96 (d, J=2.5 Hz, 1 H), 7.47 (d,J=8.5 Hz, 2 H) 7.66 (d, J=8.5 Hz, 2 H).

Conjugation of compound (9) with CP-0126 to form CP-0460 is illustratedin Synthetic Scheme 5 and described thereafter: ##STR7##

CP-0126 (100 mg, 0.08 mmol) was reacted with compound (9) (0.12 mmol,1.5 eqv) in 2 mL 95% DMF/5% 0.1 M ammonium bicarbonate containing 50 uldiisopropylethylamine, for 30 minutes, with occasional mixing. Thereaction mixture was purified in 1 injection on a Vydac 1" C-18 reversephase column at 10 ml/min, using a gradient running from 15%acetonitrile/0.1% TFA to 40% acetonitrile/0.1% TFA in 20 minutes.Appropriate fractions were lyophilized to yield 64 mg (45%) of acolorless powder (CP-0460). Laser desorption mass spectrometry:M/Z=1,802 (M+H), calculated 1,802.

As mentioned previously, for the COI to work it may need to bedissociated from the BKAn so as to allow for its intracellularpenetration. Therefore, in order to evaluate the functional activity ofa BKAn/COI heterodimer, CP-0460 was exposed to rat lung parenchymalstrips which were then challenged with arachidonic acid. This tissue isknown to contain both non-specific esterase activity as well as toconvert arachidonic acid to thromboxane (via a cyclooxygenase dependentpathway) which is then ultimately responsible for the smooth musclecontraction observed in this assay.

Using this system, the log dose ratio shifts for indomethacin andCP-0460 were found to be 0.998±0.425 and 1.029±0.042 respectivelyindicating that both indomethacin alone and CP-0460 will prevent thecontraction produced in response to exogenously applied arachidonic acidwith equal potency. BKAn's have no effect on this system in and ofthemselves. These data indicate that the COI component of BKAn/COIheterodimer is functionally active in a tissue containing bothesterolytic and cyclooxygenase activities.

Intact CP-0460 was also tested for BKAn activity using the standard ratuterus assay and the pA₂ of the CP-0460 was found to be approximately7.8. Again, CP-0460 (similarly to CP-0502) did not behave as a classicalcompetitive antagonist of bradykinin induced uterine contraction butrather as a type of "pseudo-non-competitive" antagonist, particularly athigher concentrations. This atypical behavior cannot be attributed toCOI activity per se as free indomethacin has no effect on this assay atany concentration.

Regardless of the explanation for the observed data, one skilled in theart will appreciate that, as in the other two classes of compoundsillustrated herein, pharmacologically important BKAn/COI heterodimerscan be made using a variety of appropriate linking moieties to provide afree hydroxyl and the carboxyl group (a common feature of many COIs) ofthe COI monomer to form a hydrolyzable ester based heterodimer.Compounds such as these may be used in the treatment of a variety ofinflammatory or painful conditions as well as in the treatment ofdysfunctional uterine smooth muscle activity.

While the invention has been exemplified above by the use of Ycomponents which are non-peptides, this component may equally comprisein whole or part a peptide as exemplified in the afore-mentioned Ser.No. 07/859,582 and Ser. No. 07/677,391, the entire contents of theseapplications, including the heterodimers there described, beingincorporated herein as earlier noted.

The dimers of the invention may be used in the form of conventionalpharmaceutical compositions comprising the active component and apharmaceutically acceptable carrier. Such compositions may be adaptedfor topical, oral, aerosolized, intramuscular, subcutaneous orintravenous administration. The amount of active component present insuch compositions will range from, for example, about 0.001 to 90.0% byweight depending on the application and mode of administration althoughmore or less of the active component may be used. Conventional dosageswill vary considerably on the basis of the intended application and modeof administration. Usually, however, an effective dose is in the orderof 0.1 to 1000 micrograms per kg body weight.

The scope of the invention is defined in the following claims wherein:

What is claimed is:
 1. A heterodimer of the formula:

    (BKAn)(X)(Y)

where BKAn is a bradykinin antagonist peptide; Y is selected from thegroup consisting of a neutrophil elastase inhibitor, a cyclooxygenaseinhibitor, a NK₁ receptor antagonist, and a NK₂ receptor antagonist; andX is a linking group chemically joining the BKAn and Y components andsaid linking occurs via the amino acid residue in the 0, 1, 2, 3, 5, or6 position of said BKAn.
 2. A heterodimer according to claim 1, whereinY is a neutrophil elastase inhibitor.
 3. A heterodimer according toclaim 1 wherein Y is a cyclooxygenase inhibitor.
 4. A heterodimeraccording to claim 1, wherein Y is an NK₁ receptor antagonist or NK₂receptor antagonist.
 5. A heterodimer according to claim 1, wherein X ishydrolyzable.
 6. A heterodimer according to claim 1, wherein X isnon-hydrolyzable.
 7. A heterodimer according to claim 1, wherein Xcomprises an amino acid or an amino acid analog incorporated into theBKAn.
 8. A heterodimer according to claim 1, wherein X comprises amaleimide/succinimide-based linkage.
 9. A heterodimer according to claim1, wherein X comprises a bissuccinimidoalkane.
 10. A heterodimeraccording to claim 1, wherein the BKAn comprise a cysteine residue inthe 6-position; and wherein X forms a linkage through the --S-- atom ofthe Cys⁶ sulfhydryl group of the BKAn.
 11. A heterodimer according toclaim 1, wherein Y is a neutrophil elastase inhibitor, the BKAn comprisea cysteine residue in the 6-position, and X includes a succinimide groupattached to the BKAn through the sulfur atom of the Cys⁶ sulfhydrylgroup of the BKAn.